Stable omega conopetide formulations

ABSTRACT

Disclosed are formulations effective to stabilize omega conotoxin peptide preparations at elevated temperatures. Novel omega conopeptides also form part of the invention.

FIELD OF THE INVENTION

The present invention relates to methods of preventing progression ofneuropathic pain and to formulations suitable for stabilizing omegaconopeptide peptides used in such treatment methods.

REFERENCES

Ahmad, S. N. and Miljanich, G. P., Brain Research 453:247-256 (1988).

Bennett, G. J. and Xie, Y.-K., Pain 33:87-107 (1988).

Bennett, J. P. et al., in NEUROTRANSMITTER RECEPTOR BINDING, pp. 61-89;Raven Press, New York, N.Y. (1983).

Ben-Sreti, M. M., et al., Eur. J. Pharmacol. 90:385-391 (1983).

Contreras, E., et al., Eur. J. Pharmacol. 148:463-466 (1988).

Dixon, W. J., Ann. Rev. Pharmacol. Toxicol. 20:441-462 (1976).

Gray, W., et al., Annual Review of Biochemistry 57:665-700 (1988).

Jadad, A. R., et al., Lancet 339:1367-1371 (1992).

Kenakin, T. P., in PHARMACOLOGIC ANALYSIS OF DRUGRECEPTOR INTERACTION,Raven Press, New York, N.Y. (1987).

Kim, S. H. and Chung, J. M., Pain 50:355-363 (1992).

McCleskey, E. W., et al., Proc. Natl. Acad. Sci. USA 84:4327-31 (1987).

McGeer, P. L., et al., in MOLECULAR NEUROBIOLOGY OF THE MAMMALIAN BRAIN,Plenum Press, New York, N.Y. (1987).

Nowycky, M. C., et al., Nature (London) 316:440-443 (1985).

Olivera, B., et al., Biochemistry 23:5087-5090 (1984).

Sher, E. et al., FASEB J. 5:2677-2683 (1991).

Sher, E. and Clementi, F., Neuroscience 42301-42307 (1991).

Yaksh, T. L., and Rudy, T. A., Physiol. Behav. 17:1031-1036 (1976).

Yamashiro, D., Int. J. Peptide Protein Res. 30:9-12 (1987).

BACKGROUND OF THE INVENTION

Chronic or intractable pain, as may occur in conditions such as bonedegenerative diseases, AIDS, Reflex sympathetic dystrophy (RSD), andcancer, is a debilitating condition which is treated with a variety ofanalgesic agents, and often opioid compounds, such as morphine.

Neuropathic pain is a particular type of pain that has a complex andvariable etiology. It is frequently a chronic condition attributable tocomplete or partial transection of a nerve, trauma or injury to a nerve,nerve plexus or soft tissue, or other conditions, including cancer, AIDSand idiopathic causes. Neuropathic pain is characterized by hyperalgesia(lowered pain threshold and enhanced pain perception) and by allodynia(pain from innocuous mechanical or thermal stimuli). The condition isprogressive in nature. Because the hyperesthetic component ofneuropathic pain does not respond to the same pharmaceuticalinterventions as does more generalized and acute forms of pain,development of effective longterm treatment modalities has beenproblematic.

Opioid compounds (opiates) such as morphine, while effective inproducing analgesia for many types of pain, are generally not effectivefor treating the progressive stages of neuropathic pain. Moreover, thesecompounds are known to induce tolerance in patients, so that increaseddoses are required to achieve a satisfactory analgesic effect. At highdoses, these compounds produce side effects, such as respiratorydepression, which can be life threatening. In addition, opioids canproduce physical dependence in patients. Dependence appears to berelated to the dose of opioid taken and the period of time over which itis taken by the subject. For this reason, alternate therapies for themanagement of chronic pain are widely sought after. In addition,compounds which serve as either a replacement for or as an adjunct toopioid treatment in order to decrease the dosage of analgesic compoundrequired, have utility in the treatment of pain, particularly pain ofthe chronic, intractable type.

Although calcium blocking agents, including a number of L-type calciumchannel antagonists, have been tested as adjunct therapy to morphineanalgesia, positive results are attributed to direct effects on calciumavailability, since calcium itself is known to attenuate the analgesiceffects of certain opioid compounds (Ben-Sreti, et al., 1983). EGTA, acalcium chelating agent, is effective in increasing the analgesiceffects of opioids. However, results from tests of calcium antagonistsas adjunct therapy to opioids have been contradictory; some L-typecalcium channel antagonists have been shown to increase the analgesiceffects of opioids, while others of these compounds have been shown todecrease opioid effects (Contreras, et al., 1988).

U.S. Pat. No. 5,051,403 describes the use of omega-conopeptides havingdefined binding/inhibitory properties in the treatment ofischemia-related neuronal damage. U.S. Pat. No. 5,364,842 demonstratesthe effectiveness of omega-conopeptide compositions in certain animalmodels of pain. Specifically, omega-conopeptides MVIIA and TVIA andderivatives thereof having related inhibitory and binding activitieswere demonstrated to produce analgesia in animal models of analgesia inwhich morphine is the standard positive control. Co-owned applicationU.S. Ser. No. 08/049,794 discloses that omega conopeptides also exhibitanalgesic properties in certain models of analgesia, such as neuropathicpain models of analgesia, in which morphine is not expected to producepositive results.

The present invention is based on the discovery that N-typevoltage-sensitive calcium channel (VSCC) blocking compounds, includingomega conopeptides, are effective to prevent progression of neuropathicconditions. Also disclosed are improved routes of administration forproviding relief from neuropathic pain. In addition, the presentinvention discloses stabilized conopeptide formulations that areparticularly useful in the treatment methods of the present invention.These stabilized compositions also find use in other applications inwhich prolonged administration or long-term storage of solutionscontaining conopeptides are required.

SUMMARY OF THE INVENTION

In one aspect, the invention includes a method of preventing progressionof neuropathic pain. According to the method, the subject is given anN-type voltage-sensitive sensitive calcium channel blocking compoundwhich is effective (a) to inhibit electrically stimulated contraction ofthe guinea pig ileum, and (b) to bind selectively to omega conopeptideMVIIA binding sites present in neuronal tissue. In a specificembodiment, the activities of the compound in inhibiting the guinea pigileum and in binding to the MVIIA binding site are within the ranges ofsuch activities of omega-conotoxins MVIIA and TVIA. In anotherembodiment of the invention, the neuropathic pain is characterized bynociceptor sensitization.

In a more specific embodiment, the activity of the compound to bindselectively to the omega conopeptide MVIIA binding sites is measured bya selectivity ratio of binding at the MVIIA binding site to binding at asite 2 omega conopeptide binding site. Effective compounds will have aselectivity ratio which is within the range of selectivity ratiosdetermined for omega conopeptides MVIIA/SNX-111, SNX-199, SNX-236,SNX-239 and TVIA/SNX-185.

In a preferred embodiment, the N-type calcium channel blocking compoundsare omega-conopeptides. In another preferred embodiment, the omegaconopeptide is selected from the group consisting of SEQ ID NO: 7(TVIA/SNX-185), SEQ ID NO:1 (MVIIA/SNX-111), SEQ ID NO: 30 (SNX-236),SEQ ID NO: 2 (SNX-159), SEQ ID NO: 32 (SNX-239), SEQ ID NO: 33(SNX-199), SEQ ID NO: 35 (SNX273), SEQ ID NO: 36 (SNX-279), andderivatives thereof.

Such peptides may preferably be administered by transdermaliontophoresis. In another preferred embodiment, the conopeptideformulation will include an anti-oxidant effective to prevent methionineoxidation.

In other preferred embodiments, the method of the invention will includeadministering compound by means effective to deliver compound to regionsof neuropathic pain. In one preferred embodiment, the compound isadministered by perineural application of compound. In another preferredembodiment, such administering is by topical application to a skinregion characterized by proliferation of neurite outgrowth. Such topicaladministration may further include use of a transdermal patch. Inanother embodiment, the compound is administered by subdermal injectionin a region characterized by proliferation of neurite outgrowth.

In yet another embodiment, the compound is administered by epiduralinjection. When practicing this method of the invention using an omegaconopeptide compound, the treatment method may include means forenhancing permeation of the conopeptide through meningeal membranes.Such membrane permeation enhancing means can include, for example,liposomal encapsulation of the peptide, addition of a surfactant to thecomposition, or addition of an ion-pairing agent. Also encompassed bythe invention is a membrane permeability enhancing means that includesadministering to the subject a hypertonic dosing solution effective todisrupt meningeal barriers.

In another aspect, the invention includes a stable omega conopeptideformulation. This formulation includes, in addition to an omegaconopeptide, an anti-oxidant capable of preventing methionine oxidation.In one embodiment, the anti-oxidative composition includes a carboxylicacid buffer. Generally, the buffer pH will be below about pH 6, and in apreferred embodiment, the carboxylic acid buffer is lactate bufferadjusted to a pH of about 4-4.5.

In another embodiment, the formulation includes methionine as ananti-oxidant. Generally, the formulation will be in a range below aboutpH 6. In a more specific embodiment, the methionine formulation bufferincludes 0.9% sodium chloride acidified to a pH of about 4-4.5. Theforegoing specific lactate and methionine formulations may be used aloneor in combination with each other or with other anti-oxidant orstabilizing solutions known in the art. Although such anti-oxidantformulations can be used with any omega-conopeptide that is susceptibleto oxidation, they are particularly useful for conopeptides SNX-111,SNX-178, SNX 207, and SNX-236.

The invention further includes, in a related embodiment a method ofstabilizing an omega conopeptide solution. The method includes adding toan omega conopeptide-containing solution an anti-oxidant compositioncapable of preventing methionine oxidation, as described above.

In still a further aspect, the invention includes omega-conopeptideSNX-273 having the sequence: SEQ ID NO: 35. In another aspect, theinvention includes omega conopeptide SNX-279 having the sequence SEQ IDNO: 36.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show primary sequences of several naturalomega-conopeptides, MVIIA/SNX-111 (SEQ ID NO:01), MVIIB/SNX-159 (SEQ IDNO: 02), GVIA/SNX-124 (SEQ ID NO: 03), GVIIA/SNX-178 (SEQ ID NO: 04),RVIA/SNX-182 (SEQ ID NO: 05), SVIA/SNX-157 (SEQ ID NO: 06), TVIA/SNX-185(SEQ ID NO: 07), SVIB/SNX-183 (SEQ ID NO: 08), and MVIIC/SNX-230 (SEQ IDNO: 29), and SNX-231 (SEQ ID NO: 21);

FIGS. 2A and 2B show primary sequences of analog omega-conopeptidesSNX-190 (SEQ ID NO: 09), SNX-191 (SEQ ID NO: 10), SNX-193 (SEQ ID NO:11), SNX-194 (SEQ ID NO: 12), SNX-195 (SEQ ID NO: 13), SNX-196 (SEQ IDNO: 14), SNX-197 (SEQ ID NO: 15), SNX-198 (SEQ ID NO: 16), SNX-199 (SEQID NO: 33), SNX-200 (SEQ ID NO: 17), SNX-201 (SEQ ID NO: 18), SNX-239(SEQ ID NO: 32), SNX240 (SEQ ID NO: 34), SNX-202 (SEQ ID NO: 19),SNX-207 (SEQ ID NO: 20), SNX-236 (SEQ ID NO: 30), SNX-273 ( ala¹²-SNX-111; SEQ ID NO: 35), and SNX-279 (Met(0)¹² -SNX-111; SEQ ID NO: 36)and their relationships to SNX-111 (SEQ ID NO: 01), SNX-185 (SEQ ID NO:07) or SNX-183 (SEQ ID NO: 08), where Nle indicates norleucine, andMet(0) indicates a sulfoxy-methionine substitution;

FIGS. 3A and 3B show computer-fit competitive binding curves foromega-conopeptide binding to the OCT MVIIA (SNX-111) binding site in ratbrain synaptosomes;

FIGS. 4A and 4B show computer-fit competitive binding curves foromega-conopeptide binding to the OCT MVIIC (SNX-230) binding site in ratbrain synaptosomes;

FIG. 5 shows omega-conopeptide groupings: I, MVIIA, SNX-199 (SEQ ID NO:33), MVIIB and SNX-239 (SEQ ID NO: 32), II, TVIA, SNX-207 and SNX-236,III, RVIA, SVIA, GVIIA, SVIB, MVIIC, SNX-231;

FIGS. 6A-6D show the effect of treatment with various omega-conopeptideson mechanical allodynia thresholds in rats with a painful peripheralneuropathy, where SNX-111 (6A), SNX-239 (6B), SNX-159 (6C) and SNX-230(6D) were tested at the doses indicated;

FIGS. 7 (A,B) shows dose response curves of effects ofomega-conopeptides SNX-111 (7A) and SNX-239 (7B) derived from the dataillustrated in FIGS. 6A and 6B, respectively, in a rat model ofneuropathic pain;

FIGS. 8A and 8B show dose-dependent blockade of mechanical allodynia bySNX-273 (8A) and SNX-279 (8B) in comparison to saline and SNX-111 atvarious times post treatment, where asterisks indicate statisticallysignificant differences between treatment and saline (p<0.05, Student'st test); and

FIG. 9 shows analgesic efficacy of an SNX-111 formulation combiningmethionine in lactate buffer.

DETAILED DESCRIPTION OF THE INVENTION I. N-type Voltage-SensitiveCalcium Channel Blocking Compounds

Voltage-gated calcium channels are present in neurons, and in cardiac,smooth, and skeletal muscle and other excitable cells. These channelsare known to be involved in membrane excitability, muscle contraction,and cellular secretion, such as in exocytotic synaptic transmission(McCleskey, et al., 1987). In neuronal cells, voltage-gated calciumchannels have been classified by their electrophysiological as well asby their biochemical (binding) properties.

Calcium channels are generally classified according to theirelectrophysiological properties as Low-voltage-activated (LVA) orHigh-voltage-activated (HVA) channels. HVA channels are currently knownto comprise at least three groups of channels, known as L-, N- andP-type channels (Nowycky, et al., 1985; Sher, et al., 1991). Thesechannels have been distinguished one from another structurally andelectrophysiologically as well as biochemically on the basis of theirpharmacology and ligand binding properties. Thus, dihydropyridines,diphenylalkylamines and piperidines bind to the alpha, subunit of theL-type calcium channel and block a proportion of HVA calcium currents inneuronal tissue, which are termed L-type calcium currents.

N- or omega- type HVA calcium channels are distinguishable from othercalcium channels by their sensitivity to omega conotoxins (omegaconopeptides). Such channels are insensitive to dihydropyridinecompounds, such as L-type calcium channel blockers nimodipine andnifedipine (Sher, et al., 1991; Sher and Clementi, 1991).

A. Omega-Conopeptides Omega-conopeptides are components of peptidetoxins produced by marine snails of the genus Conus, and which act ascalcium channel blockers (Gray, et al., 1988). About 500 species of conesnails in the Conus genus have been identified, and a variety ofomega-conopeptides from several of these species have been isolated. Theprimary sequences of eight naturally-occurring omega-conopeptides areshown in FIG. 1, where SNX-231 is an alternative form of MVIIC/SNX-230.Conventional letter initials are used for the amino acid residues, and Xrepresents 4-hydroxyproline, also abbreviated 4Hyp. All of the peptidesshown in the figure are amidated at their C-termini.

The peptides shown in FIG. 1 are identified by names which are commonlyassociated with either the naturally occurring peptide (single letterfollowed by a Roman numeral followed by a single letter), and by asynthetic designation (SNX-plus numeral). Either or both of thesedesignations will be used interchangeably throughout the specification.For example, the peptide whose sequence is designated MVIIA/SNX-111 willbe referred to herein as OCT MVIIA, or alternatively, SNX-111, thelatter to signify that the compound is synthetic in origin. Syntheticand naturally occurring peptides having the same sequence behavesubstantially identically in the assays and methods of treatment of theinvention. The OCT MVIIA (SNX-111) and OCT GVIA (SNX-124) peptides alsohave the common names CmTx and CgTx, respectively. All of theomega-conopeptides have three disulfide linkages connecting cysteineresidues 1 and 4, 2 and 5, and 3 and 6, as indicated for the MVIIApeptide in FIG. 2A. FIGS. 2A and 2B shows analogs or derivatives ofnatural OCT MVIIA, OCT TVIA, and OCT SVIB peptides which have beensynthesized and tested in accordance with the invention. Standard singleamino acid code letters are used in the figure; X=hydroxyproline;Nle=norleucine; Met(O)=sulfoxy-methionine; NH₂ group at the C terminusindicates that the peptide is C-terminal amidated; G--OH indicatestermination in an unmodified glycine residue.

B. Preparation of Omega Conopeptides

This section describes the synthesis, by solid phase methods, of severalnaturally occurring omega conotoxin (OCT) peptides and additionalomega-conopeptides which are used in the present invention.

Omega-conopeptides, such as those shown in FIGS. 1 and 2, can besynthesized by conventional solid phase methods, such as have beendescribed (Olivera, et al., 1984). Briefly, N-alpha-protected amino acidanhydrides are prepared in crystallized form or prepared freshly insolution and used for successive amino acid addition at the N-terminus.At each residue addition, the growing peptide (on a solid support) isacid treated to remove the N-alpha-protective group, washed severaltimes to remove residual acid and to promote accessibility of thepeptide terminus to the reaction medium. The peptide is then reactedwith an activated N-protected amino acid symmetrical anhydride, and thesolid support is washed. At each residue-addition step, the amino acidaddition reaction may be repeated for a total of two or three separateaddition reactions, to increase the percent of growing peptide moleculeswhich are reacted. Typically, 1-2 reaction cycles are used for the firsttwelve residue additions, and 2-3 reaction cycles for the remainingresidues.

After completing the growing peptide chains, the protected peptide resinis treated with liquid hydrofluoric acid to deblock and release thepeptides from the support. For preparing an amidated peptide, the resinsupport used in the synthesis is selected to supply a C-terminal amide,after peptide cleavage from the resin. After removal of the hydrogenfluoride, the peptide is extracted into 1M acetic acid solution andlyophilized. The three disulfide linkages in the peptides may be formedby air oxidation in the presence of dithiothreitol (DTT) or optionallyother thiol containing compounds (e.g., cysteine, glutathione),according to procedures detailed in Example 1.

The peptide can be isolated by an initial separation by gel filtration,to remove peptide dimers and higher molecular weight polymers, and alsoto remove undesired salts, such as guanidine hydrochloride, used in theoxidation reaction. The partially purified peptide is further purifiedby preparative HPLC chromatography, and the purity and identity of thepeptide confirmed by amino acid composition analysis, mass spectrometryand by analytical HPLC in two different solvent systems.

C. Stable Omega-conopeptide Formulations

Dilute solutions of omega-conopeptides are generally unstable insolution, as evidenced by oxidation of methionine residues and reductionor loss of biological activity. In accordance with an important aspectof the present invention, it has been discovered that omega-conopeptidescan be significantly stabilized in solution by preventing oxidation ofmethionine residues present in the peptide structure. In particular,SNX-111, which contains a methionine at position 12, is approximately10-fold less potent in binding to omega-conopeptide MVIIA binding sites,when its methionine is present in the sulfoxy form.

In experiments carried out in support of the present invention, it hasbeen found that SNX-111 oxidation can be prevented by addition oflactate buffer to the composition. More particularly buffers containing150 mM lactate buffer, pH 4-4.5 improve stability of the compoundconsiderably. It is known that solutions of SNX-111 in which the peptideconcentration is less than about 0.1 mg/ml oxidize rapidly whendissolved in water, saline, or any of a number of buffers used in theart of peptide chemistry. It is a discovery of the present inventionthat solutions of SNX-111 ranging from 0.01-0.1 mg/ml are stable at 45°C. for weeks, when stabilized with lactate (150 mM, pH 4-4.5). Inaddition, buffers containing 50 μg/ml methionine are also effective instabilizing SNX-111. Here, either 150 mM lactate buffer or acidifiedsaline (pH 4-4.5) can be used to buffer the solution.

The foregoing stabilization method and formulation will find particularuse in preventing oxidation of those compounds containing methionineresidues. With reference to FIGS. 1 and 2, such compounds includeSNX-111, SNX-178, SNX-190, SNX-191, SNX-193, SNX-194, SNX-197, SNX-198,SNX-199, SNX-200, SNX-202, SNX-207, SNX-236, SNX-237, SNX-239, SNX-240;however, it is appreciated that the formulation buffer conditions may beused with peptides that lack methionine, as well.

Formulations which incorporate the components or principles discussedabove may be used in a number of pharmaceutical applications related toomega conopeptide administration. Solutions of peptides provided invials may be stored under the acidic formulation conditions, prior todilution into a pharmaceutical excipient suitable for parenteraladministration. Solutions used for slow infusion may also be preservedin this manner. The solution may be administered directly or neutralizedprior to administration, according to the particular route ofadministration in which the formulation is used. For example, directintrathecal administration of approximately 10 μl of SNX-111 in lactatebuffer (150 mM, pH 4-4.5) has been used in treating rats, withoutnoticeable untoward effects. For administration to the bloodstream theacidified physiological saline formulation may prove preferable, oreither preparation may be neutralized by dilution in a neutralizingphysiological excipient, such as a phosphate buffered saline, just priorto administration.

D. In vitro Properties of N-type VSCC Blocking compounds

This section describes some of the in vitro properties of N-type VSCCblocking compounds, as exemplified by a specific group of omegaconopeptides, namely those omega conopeptides that, like omegaconopeptide MVIIA, exhibit high affinity binding to the MVIIA (site 1)binding site and relatively low affinity binding to the SVIB (site 2)omega conopeptide binding site, as discussed below.

1. Calcium-Channel Antagonist Activity. Omega conotoxins bind to aspecific population of binding sites, present mainly in neuronal tissue.Dihydropyridines and other L-type channel blockers do not displace omegaconotoxin binding, nor do omega conotoxins displace binding of suchL-channel specific ligands to L-type calcium channels. Theseobservations indicate that L-type calcium channel blockers and N-typecalcium channel blockers act at distinct channels. Unlike L-type calciumchannels, N-type or omega channels are found predominantly, although notexclusively, in nervous tissue (Sher, et al., 1991).

Inhibition (blockade) of N-type or omega HVA neuronal calcium channelscan be conveniently measured in an isolated cell system, such as themouse neuroblastoma cell line, strain N1E115 or the human neuroblastomacell line IMR32, as described in U.S Pat. No. 5,364,842. As demonstratedtherein, N-type calcium currents are blocked by omega conopeptide MVIIA,but not by dihydropyridines.

2. Specific, High Affinity Binding to OCT Receptors. Omega-conopeptideshave been shown, in accordance with the invention, to bind with highaffinity to specific binding site(s) in neuronal cells. In accordancewith the selectivity of the compound, the binding affinity can becharacterized either by the binding constant of the compound for thehigh-affinity MVIIA (SNX-111) binding site, also referred to as "site 1" herein, or the binding constant of the compound for the high-affinitySVIB (SNX-183) or the MVIIC (SNX-230) binding site, also referred to as"site 2 " herein. Characteristics of these two distinct OCT bindingsites are summarized below. In some cases, it is useful to characterizeomega-conopeptides according to the ratio of their binding constantsmeasured for binding to neuronal-cell MVIIA (SNX-111)-specific bindingsite 1 and SVIB (SNX-183)- or MVIIC (SNX-230)-specific binding site 2.

Binding to the OCT MVIIA binding site in neuronal tissue can bedemonstrated in a variety of cell types and synaptosomal cell fractions.One preferred synaptosomal fraction is a mammalian brain synaptosomalmembrane preparation, such as the rat brain synaptosome preparationdescribed in U.S. Pat. No. 5,364,842. The binding constant of a compoundfor the MVIIA binding site is typically determined by competitivedisplacement of radiolabeled OCT MVIIA (SNX-111) from the synaptosomalpreparation, as follows.

The binding constant (K_(d)) of the MVIIA (SNX-111) peptide for thesynaptosomal membranes is determined by a saturation binding method inwhich increasing quantities of radiolabeled peptide are added to thesynaptosomal membranes, and the amount of labeled material bound at eachconcentration is determined (Example 2A). The appropriate bindingequation describing the concentration of bound ligand as a function ofthe total ligand in equilibrium is fitted to the data to calculate theB_(max) (the concentration of binding sites on the synaptosomes), andthe K_(d) (which is approximately the concentration of the ligandrequired for half saturation of binding sites).

Using conventional Scatchard analysis, a K_(d) binding value ofapproximately 10 pM is obtained for omega conopeptide MVIIA. SimilarlyK_(d) 'S were determined for binding of radiolabelled SVIB (SNX-183) andMVIIC (SNX-230) to binding sites in synaptosomal membranes.

To determine the binding constant of a test N-type VSCC blockingcompound for an OCT binding site, the test compound is added, atincreasing concentrations, to a membrane preparation, such as asynaptosome preparation, in the presence of a standard concentration ofa radiolabeled OCT which exhibits reversible binding, such as OCT MVIIA(SNX-111). The synaptosomal material is then rapidly filtered, washedand assayed for bound radiolabel. The binding constant (K_(i)) of thetest compound is determined from computer-fit competitive bindingcurves, such as shown in FIGS. 3A and 3B for MVIIA (SNX-111) peptide, todetermine first the IC₅₀ value of the compound, i.e., the concentrationwhich gives 50% displacement of labeled MVIIA peptide. A K_(i) isdetermined according to standard methods from the K_(d) value of OCTMVIIA and the IC₅₀ value of the compound, as detailed in Example 2. Arelative potency value can also be calculated from this information, asillustrated. Like the K_(i) value, this value allows comparisons betweenassays performed under slightly differing conditions or at differenttimes. While the specific value for a particular compound may vary frompreparation to preparation, the rank order of binding affinities amongthe compounds should remain essentially unchanged. Thus the potency of aparticular compound can be compared to standard compounds within a givenpreparation, to determine whether the test compound within a potencyrange considered useful in the methods of the invention, as discussedbelow.

Calculated IC₅₀ values for a number of omega-conopeptides for binding ofOCT MVIIA (SNX-111) to a rat synaptosomal preparation are given inTable 1. The compounds are arranged in order of increasing IC₅₀ values.

                  TABLE 1                                                         ______________________________________                                        COMPETITION OF .sup.125 I-MVIIA (SNX-111)                                     BINDING BY OCT PEPTIDES                                                                      IC.sub.50 (nM)                                                 ______________________________________                                        SNX-207          .007                                                         SNX-194          .008                                                         SNX-195          .009                                                         MVIIA (SNX-111)  .010                                                         SNX-190          .021                                                         SNX-236          .030                                                         SNX-200          .039                                                         SNX-201          .046                                                         SNX-202          .046                                                         SNX-193          .070                                                         SNX-194          .090                                                         SNX-239          .090                                                         MVIIC (SNX-230)  .32                                                          MVIIB (SNX-159)  .101                                                         GVIA (SNX-124)   .134                                                         SNX-198          .160                                                         SNX-191          .165                                                         TVIA (SNX-185)   .228                                                         SNX-196          .426                                                         RVIA (SNX-182)   .893                                                         SVIB (SNX-183)   1.5                                                          GVIIA (SNX-178)  3.70                                                         SNX-197          11.3                                                         SVIA (SNX-157)   1460.                                                        ______________________________________                                    

Similarly, IC₅₀ and K_(i) values for compound binding to the SVIB(SNX-183) binding site can be calculated, as above, by determining theK_(d) of labeled OCT SVIB (SNX-183) or OCT MVIIC (SNX-230) binding to asynaptosome preparation, then using competitive displacement of thelabeled compound by the test compound, to determine the IC₅₀ and K_(i)or relative potency values of the test compound. FIGS. 4A and 4B showcomputer-fit competitive binding curves for several omega-conopeptideswhose binding to the SVIB (SNX-183) and/or MVIIC (SNX-230) binding siteswas examined. From these curves, IC₅₀ values were determined as above.

Tables 2 and 3 list the relative potencies for binding of variousomega-conopeptides to the site 1 and site 2 binding sites, and show theratio of Ki or IC₅₀ values determined for binding of each compound tothe sites.

                  TABLE 2                                                         ______________________________________                                        SELECTIVITY OF CONOPEPTIDES FOR SITE 1 AND SITE 2                                    Ki (nM) for competition.sup.a                                                                   Selectivity.sup.b                                           with:             for:                                                 Compound  .sup.125 I!-SNX-111                                                                       .sup.125 I!-SNX-230                                                                      site 1                                                                              site 2                                 ______________________________________                                        SNX-111  0.002       150         75,000                                                                              1                                      SNX-183  0.43        6              14 1                                      SNX-230  0.20        0.03           1  7                                      ______________________________________                                         .sup.a Ki values were derived from analysis of competitive binding            performed as described in FIG. 1.                                             .sup.b Selectivity is expressed as the ratio of the Ki value determined       for competition with highaffinity  .sup.125 ISNX-230 binding divided by       the Ki value for competition with  .sup.125 ISNX-111 binding.            

                  TABLE 3                                                         ______________________________________                                        SELECTIVITY OF CONOPEPTIDES FOR SITE 1 AND SITE 2                                    IC.sub.50 (nM) for competition                                                                 Selectivity.sup.a                                            with:            for:                                                  Compound  .sup.125 I!-SNX-111                                                                      .sup.125 I!-SNX-230                                                                      site 1 site 2                                 ______________________________________                                        SNX-199  0.09       5,000       56,000 1                                      SNX-236  0.03       1,500       50,000 1                                      SNX-239  0.09       10,000      111,000                                                                              1                                      ______________________________________                                         .sup.a Selectivitty is expressed as the ratio of the IC.sub.50 value          determined for competition with  .sup.125 ISNX-230 binding divided by the     IC.sub.50 value for competition with  .sup.125 ISNX-111 binding.         

3. Selective Inhibition of Neurotransmitter Release. Omega-conopeptidesinhibit neurotransmitter release in various regions of the nervoussystem. As shown below, such inhibition varies according to theneurotransmitter, the omega-conopeptide, and the region studied.Neurotransmitters which can be measured, in accordance with variousaspects of the invention, include, but are not limited to dopamine,norepinephrine, acetylcholine, GABA, glutamate, and a number of peptideneurotransmitters, such as calcitonin gene-related peptide (McGeer, etal., 1987).

Quantitation of release and inhibition thereof is determined bysensitive detection methods, also known in the art, including directdetection of release of endogenous stores by HPLC or specificradioimmunoassay (RIA), and detection of release of pre-loaded, labeledcompound. Alternatively, or in addition, detection of release may beachieved using a number of indirect assays, exemplified by theelectrophysiological studies described above, in which whole tissueresponse to electrical or chemical stimulation is measured.

Inhibition of release of the neurotransmitter norepinephrine fromneuronal cells can be assayed in a number of systems known in the art,and, particularly, in mammalian brain hippocampal slices by standardmethods, such as detailed in U.S. Pat. No. 5,364,842. According to thedata shown therein, SNX-111 inhibits release of norepinephrine with highpotency (IC₅₀ ≈1 nM) but only partially (approx. 60%). SNX-183 is muchless potent (IC₅₀ ≈180 nM) but the inhibition is substantially 100%.SNX-230 also inhibits release completely, but in a biphasic manner,inhibiting approximately 50% with high potency (IC₅₀ =0.02 nM) and 50%with much lower potency (IC₅₀ =65 nM). These results suggest that suchnorepinephrine release is mediated by at least two distinct subtypes ofpresynaptic calcium channels, one of which corresponds to the site 1receptor identified by high affinity for SNX-111 and the other to thesite 2 receptor recognized preferentially by SNX-230.

Further means of measuring inhibition of neuronal transmitter releaseare isolated tissue assays, such as atrial strip, aorta, vas deferensand guinea pig ileum assays, in which the response to a stimulus,usually an electrical stimulus, is correlated to the amount ofneurotransmitter released from neurons innervating the tissue (Kenakin,1987). In the guinea pig ileum, inhibition of electrically stimulatedcontractions is correlated with inhibition of acetylcholine release, asdemonstrated by the ability of cholinergic agonists to overcome suchinhibition. Example 3 describes the preparation and assay in detail.Table 4 shows the IC₅₀ values for various omega-conopeptides oncontraction of guinea pig ileum in response to electrical stimulation.

                  TABLE 4                                                         ______________________________________                                        EFFECTS OF CONOPEPTIDES ON ELECTRICALLY                                       STIMULATED CONTRACTION OF GUINEA PIG ILEUM                                    Compound      ID.sub.50 (nM)                                                  ______________________________________                                        SNX-111       13                                                              SNX-185       29                                                              SNX-183       91                                                              SNX-157       >100                                                            ______________________________________                                    

II. Treatment of Neuropathic Pain

U.S. Pat. No. 5,364,842 describes analgesic properties of selectedomega-conopeptides. This discovery was extended to include treatment ofneuropathic pain as disclosed in co-owned U.S. patent application Ser.No. 08/049,794, filed Apr. 15, 1993, and incorporated herein byreference. It is the discovery of the present invention that progressionof neuropathic pain can be retarded in a subject exhibiting early stagesymptoms of neuropathic pain, particularly by providing localizeddelivery of N-type VSCC blocking compounds to the neuropathic site.

A. Prevention of Progression of Neuropathic Pain

In general, while brain pathways governing the perception of pain arestill incompletely understood, sensory afferent synaptic connections tothe spinal cord, termed "nociceptive pathways" have been documented insome detail. In the first leg of such pathways, C- and A-fibers whichproject from peripheral sites to the spinal cord carry nociceptivesignals. Polysynaptic junctions in the dorsal horn of the spinal cordare involved in nociceptive processing and in the relay of sensations ofpain to various regions of the brain. Analgesia, or the reduction ofpain perception, can be effected directly by decreasing transmissionalong such nociceptive pathways. Modulation of nociception is achievedby neural pathways descending from the cortex and hypothalamus to themesencephalic central grey region, medullary reticular formation, andultimately, the dorsal horn of the spinal cord. Analgesic opiates arethought to act by mimicking the effects of endorphin or enkephalinpeptide-containing neurons, which synapse presynaptically at the C- orA-fiber terminal and which, when they fire, inhibit release ofneurotransmitters, including substance P, excitatory amino acids andcalcitonin gene-related peptide (CGRP). Descending pathways from thebrain are also inhibitory on C- and A-fiber firing.

While neuropathic pain is known to have a number of underlyingetiologies, it is characterized by a distinct set of symptoms. Asdescribed in greater detail below, these can include enhancedsensitivity to innocuous thermal-mechanical stimuli, abnormalsensitivity to noxious stimuli, tenderness, and spontaneous burningpain. Neuropathic pain is also progressive in nature, in that itgenerally worsens over time. Known treatment methods treat the symptomswithout necessarily lessening the underlying pathology.

Although the present invention should not be limited by a particulartheory of underlying mechanism, it has been observed that chronicneuropathic pain in humans is accompanied by changes in excitability ofprimary nociceptive afferents. This phenomenon is known as "nociceptorsensitization" and is characterized by increased excitability of theafferents to normally subthreshold stimuli. It is the discovery of thepresent invention that treatment with omega conopeptides and, moregenerally, N-type VSCC blockers, inhibit this phenomenon.

In accordance with the present invention, omega-conopeptides useful intreating neuropathic pain are also effective in preventing itsprogression. Such "neuropatholytic" omega-conopeptides may bedistinguished and selected by their abilities (a) to inhibitvoltage-gated calcium channels selectively in neuronal tissue, asevidenced by the peptide's ability to inhibit electrically stimulatedcontraction of the guinea pig ileum, and (b) to bind to omegaconopeptide MVIIA binding sites present in neuronal tissue. Such bindingto omega conopeptide MVIIA binding sites (site 1, as described herein)is selective, as evidenced by relatively high binding affinity at suchsites, as compared to binding at an omega conopeptide site 2 (describedherein as a high affinity binding site for SNX-230 or SNX-183). Suchselectivity can be measured by the selectivity ratio illustrated inTables 2 and 3, above.

Moreover, in accordance with the invention, it has been found thatneuropatholytic omega-conopeptides are effective as analgesic agentsboth in traditional opiate-sensitive models of nociceptive pain, such asthe Rat Tail-Flick test or the rat hind-paw formalin test, as well as inopiate-resistant models of pain, such as chronic constriction injurymodels of neuropathic pain.

B. Treatment Modes for Preventing Progression of Neuropathic Pain

While the present invention is not intended to be limited by adherenceto any particular theory of underlying mechanism, one theory which isconsistent with the invention is that acute nociception leads toalteration of signal transduction mechanisms in pain pathways. Underthis theory, interference with such signal transduction by blockade ofN-type VSCC's prevents this alteration.

It is a discovery of the present invention that N-type calcium channelblocking omega-conopeptides are effective to prevent development of orprogression of neuropathic pain, when administered perineurally toaffected skin regions characterized by proliferation of neuriteoutgrowth subsequent to nerve damage.

Perineural administration can be by topical means, either directly orwith the use of a transdermal applicator. Alternatively, perineuraladministration may be effected by subdermal injection. The resultingblockade of calcium channels reduces the heightened sensation producedby transmission through the neurite proliferation. Perineural deliverymay also be effected by forming a cuff around the damaged nerve,preferably of a biodegradable matrix which includes a therapeutic N-typecalcium channel blocking compound. Alternatively or in addition, thetherapeutic compound can be placed in close proximity to the damagednerve by conjugating the compound to or coating the compound on a nervesplint designed for repairing damaged nerves. Examples of such nervesplints are provided by U.S. Pat. Nos. 4,534,349 and 4,920,962.

Perineural delivery may also be effected by incorporating N-type calciumchannel blocking compounds into suture materials, and using thesematerials to suture damaged tissues. This method is particularly usefulfor delivery of compound in areas where it is desirable to provide forinhibition of progression of neuropathy concomitant to tissue damage.U.S. Pat. No. 5,308,889 describes a collagen-based suture material thatmay be suitable for use for therapeutic delivery of N-type calciumchannel blocking compounds.

One particularly useful means for delivering compound to perineuralsites is transdermal delivery. This form of delivery can be effectedaccording to methods known in the art. Generally, transdermal deliveryinvolves the use of a transdermal "patch" which allows for slow deliveryof compound to a selected skin region. Although such patches aregenerally used to provide systemic delivery of compound, in the contextof the present invention, such site-directed delivery can be expected toprovide increased concentration of compound in selected regions ofneurite proliferation. Examples of transdermal patch delivery systemsare provided by U.S. Pat. No. 4,655,766 (fluid-imbibing osmoticallydriven system), and U.S. Pat. No. 5,004,610 (rate controlled transdermaldelivery system).

For transdermal delivery of peptides, such as omega-conopeptidesdescribed herein, transdermal delivery may preferably be carried outusing iontophoretic methods, such as described in U.S. Pat. No.5,032,109 (electrolytic transdermal delivery system), and in U.S. Pat.No. 5,314,502 (electrically powered iontophoretic delivery device).

For transdermal delivery, it may be desirable to include permeationenhancing substances, such as fat soluble substances (e.g., aliphaticcarboxylic acids, aliphatic alcohols), or water soluble substances(e.g., alkane polyols such as ethylene glycol, 1,3-propanediol,glycerol, propylene glycol, and the like). In addition, as described inU.S. Pat. No. 5,362,497, a "super water-absorbent resin" may be added totransdermal formulations to further enhance transdermal delivery.Examples of such resins include, but are not limited to, polyacrylates,saponified vinyl acetate-acrylic acid ester copolymers, cross-linkedpolyvinyl alcohol-maleic anhydride copolymers, saponifiedpolyacrylonitrile graft polymers, starch acrylic acid graft polymers,and the like. Such formulations may be provided as occluded dressings tothe region of interest, or may be provided in one or more of thetransdermal patch configurations described above.

For delayed release, the N-type calcium channel blocking compound may beincluded in a pharmaceutical composition for formulated for slowrelease, such as in microcapsules formed from biocompatible polymers orin liposomal carrier systems according to methods known in the art.

For continuous release of peptides, the peptide may be covalentlyconjugated to a water soluble polymer, such as a polylactide orbiodegradable hydrogel derived from an amphipathic block copolymer, asdescribed in U.S. Pat. No. 5,320,840. Collagen-based matrix implants,such as described in U.S. Pat. No. 5,024,841, are also useful forsustained delivery of peptide therapeutics. Also useful, particularlyfor subdermal slow-release delivery to perineural regions, is acomposition that includes a biodegradable polymer that is self-curingand that forms an implant in situ, after delivery in liquid form. Such acomposition is described, for example in U.S. Pat. No. 5,278,202.

The method of the invention also includes local administration ofcompound to those regions of the spinal cord, such as to dorsal hornregions at affected vertebral levels, where polysynaptic relay of painsensation occurs. This type of local application can be effected byintrathecal administration, as described in above-referenced co-pendingapplication U.S. Ser. No. 08/049,794. Intrathecal administrationdelivers compound directly to the sub-arachnoid space containingcerebral spinal fluid (CSF). While effective, this method requiresprecise technical expertise to ensure delivery to the correct spot.

It is appreciated that delivery to spinal cord regions can also be byepidural injection to a region of the spinal cord exterior to thearachnoid membrane. It is further appreciated that when the N-typevoltage-sensitive calcium channel (VSCC) blocking compound is an omegaconopeptide, it may be advantageous to add to the conopeptidecomposition means for enhancing permeation of the conopeptide throughmeningeal membranes. Such means are known in the art and includeliposomal encapsulation, or addition of a surfactant or an ion-pairingagent to the peptide composition.

Alternatively, or in addition, increased arachnoid membrane permeationcan be effected by administering a hypertonic dosing solution effectiveto increase permeability of meningeal barriers.

N-type calcium channel blocking compounds can also be administered byslow infusion. This method is particularly useful, when administrationis via the intrathecal or epidural routes mentioned above. Known in theart are a number of implantable or body-mountable pumps useful indelivering compound at a regulated rate. One such pump described in U.S.Pat. No. 4,619,652 is a body-mountable pump that can be used to delivercompound at a tonic flow rate or at periodic pulses. An injection sitedirectly beneath the pump is provided to deliver compound to the area ofneed, for example, to the perineural region.

In other treatment methods, N-type calcium channel blocking compoundsmay be given orally or by nasal insufflation, according to methods knownin the art. For administration of peptides, it may be desirable toincorporate such peptides into microcapsules suitable for oral or nasaldelivery, according to methods known in the art.

Efficacy of the foregoing methods of treatment are conveniently measuredin the allodynia model described in Example 4 herein. In this model ofperipheral neuropathy, it has been observed that neuropathic painprogressively increases over days 1-7 after the nerve insult, with aplateau of pain response thereafter. To measure ability of a compound toprevent such progression, compound is given just before, during or afternerve insult, and threshold pain responses are measured on days 1-7thereafter. Prevention of progression of neuropathic pain is observedwhen there is a heightened threshold to pain stimulus during days 1-7 orwhere there is a prolongation of time to plateau, as compared to controlanimals. Efficacious dosages and formulations determined in this modelare extrapolated to equivalent large animal and human dosages, accordingto methods known in the art.

C. Therapeutic Indications

As stated above, neuropathic pain may result from a number of separateetiologies. Generally, progression of such pain may be treated accordingto any of the methods described herein. However, in many cases it willbe preferable to treat the pain in a manner that addresses its specificsource. For example, when the pain is traceable to injury of aparticular nerve fiber, it may be appropriate to treat such pain eitherby perineural application of compound to the affected nerve or by dermalapplication of compound to the affected region.

While a discussion of specific formulations and modes of delivery can befound in foregoing Section II.B. and in Section II.F, below, thissection sets forth some exemplary indications for which treatmentaccording to the methods of the present invention may have particulartherapeutic utility. The indications described below are by no meansexhaustive, but are presented to illustrate some of the varioustherapeutic situations in which neuropatholytic N-type calcium channelblocking compounds can be used.

1. Ophthalmic indications. The eye is a heavily innervated organ. Thecornea in particular is heavily innervated with C-fibers, containing anestimated 3-4000 fiber endings per mm² compared to an estimated 3-600fiber endings per mm² of skin. Injury of the nerve fibers can lead toneuropathic pain of ophthalmic origin. In accordance with the presentinvention, the eye may be treated with N-channel blocking compounds andparticularly with omegaconopeptides, to prevent progression ofneuropathy. Application of compound may be achieved by topicaladministration to the eye, or, in more severe cases, by means ofsuprachoroidal administration. Such administration may be convenientlyachieved by providing a suprachoroidal implant which includes, forexample, omega conopeptide SNX-111. U.S. Pat. No. 5,164,188 describes abiodegradable implant that is suitable for chronic and controlledadministration of compound to the suprachoroidal space. Chronic,implanted therapeutics are also indicated after ophthalmic surgery, suchas after surgery for detached retina or macular holes, where nervedamage may result.

2. Dental indications. Delivery of N-channel blocking compounds toregions of dental repair, such as endodontic repair concomitant to aroot canal procedure, may be desirable as a means of preventingprogression of dental neuropathy. Here, the therapeutic compound may beincluded in or added to one or more of the polymer based materials orresins inserted into the root canal after removing the pulp from theregion, in accordance with standard techniques known in the art.

3. Burn injury. Burn injuries are characterized by primary hyperalgesiato thermal and mechanical stimuli. In accordance with the principlesdiscussed above, treatment of burned regions with N-type calcium channelblocking compounds may reduce progression of the hyperalgesic responseby interfering with signal transduction mechanisms of nociceptor sensoryreceptors. In this embodiment of the invention, the therapeutic compoundcan be applied directly to the affected regions, or can be applied in aformulation that includes a protective biopolymer matrix, such as anartificial skin matrix.

4. Reflex sympathetic dystrophy (RSD). RSD is thought to be due toabnormalities in the peripheral nervous system, and more particularly,to sensitization of cutaneous somatosensory afferents. Sympatheticoutflow is thought to activate foci of ectopic neural hyperexcitability.Treatment of this condition to prevent its progression may be effectedby any of the various dermal (topical) or subdermal routes of deliverydiscussed above. Perineural delivery may also be indicated for thiscondition.

5. Post-herpetic neuralgia. Post-herpetic neuralgia is characterized, inits acute phase, by intraneural inflammation which can cause damage toprimary afferent axons. This damage may result in abnormal sensitivityto cutaneous stimuli. In general, the mode of treatment to preventprogression of abnormal sensitivity will depend on the location in thebody of the affected nerve(s). Perineural or topical delivery oftherapeutic N-type calcium channel blocking compound is indicated inthis condition.

6. Diabetic neuropathy. Neuropathy of primary afferent axons in longnerves is found in diabetic patients. This results in the dying-back andattempted regeneration of distal tips of primary afferent axons of thesenerves. Nociceptor sensitization may ensue. Such sensitization and itsprogression may be treated according to one or more of the treatmentmethods described herein. In particular, perineural or topicalapplication of therapeutic compound will be indicated, in accord withthe location of the affected nerve and nerve beds.

7. Arthritis. Arthritis is characterized by enhanced sensation of painvia articular afferents. N-type calcium channel blocking compounds findutility in treatment of such pain according to the principles set forthin the present invention. Generally, in treating articular afferents,therapeutic compound will be administered perineurally, in the vicinityof the affected joint.

D. Omega-Conopeptides

In accordance with this invention, neuropatholytic omega-conopeptidesconform to certain physical and chemical constraints, as describedbelow. Generally, omega-conopeptides useful in the treatment methods arethose which are 25-35 amino acids in length and which have threedisulfide bonds at specified positions along their length.

Based on a sequence homology analysis of the peptides whose fullsequences are known (FIG. 1), the naturally occurring activeomega-conopeptides were grouped into distinct groups I and II, each withinternal homologies distinct to that group, as can be appreciated fromFIG. 5. Group I includes active omega-conopeptides MVIIA (SNX-111),MVIIB (SNX-159) and SNX-239, which possess binding constants to theMVIIA site within the range of compounds showing activity in treatingpain. Group II includes TVIA (SNX-185), SNX-207 and SNX-236, which alsopossess binding constants in the range of compounds for analgesia. Athird group includes inactive peptides SNX-231, and SVIA (SNX-157) andomega-conopeptides whose binding activities for the MVIIA site onneuronal membranes and/or activity in norepinephrine inhibition areoutside the range of active compounds.

The three groups of omega-conopeptides are arranged in FIG. 5 with theirsix Cys residues aligned, which places these residues at positions 1, 8,15, 16, 20, and 28. To make this alignment, gaps were introduced at thepositions shown in the three groups. In the analysis below, these gapsretain the assigned number shown in FIG. 5, even though they representamino acid deletions in the respective groups of activeomega-conopeptides.

Sequence variation in the peptides, based on primary structure alone,was analyzed by adopting the following constraints:

1. The peptides in both active groups (I and II) include the Cysresidues at position 1, 8, 15, 16, 20, and 28. Other Cys residues couldbe substituted at the positions indicated below only if they areselectively protected during oxidation of the peptide to form the threedisulfide linkages.

2. The peptides in the active groups include three disulfide linkagesconnecting the Cys residues at positions 1 and 16, 8 and 20, and 15 and28. As described above, the disulfide bridges are formed by airoxidation of the full sequence peptide in the presence of DTT. Theability of the peptide to form the three desired disulfide linkageswould therefore require that the peptide, prior to disulfide bridging,be able to adopt a conformation which allows the three selectedlinkages, with or without the Cys protecting-group strategy discussedabove. This constraint would thus exclude amino acid variations whichprevent or otherwise hinder the formation of the three selected bridges.

Constraints 1 and 2 preserve the basic conformation of theomega-conopeptides imposed by the three disulfide bridges.

3. Within Group I, the amino acid variations which occur at the sixnon-conserved residues are allowed, including peptides in which thecarboxy terminus is amidated or has a free acid form. That is, the firstgroup compound derivatives include the peptide structures having theform: SEQ ID NO: 22-X₁ -SEQ ID NO: 23-X₂ -X₃ -X₄ -X₅ -SEQ ID NO: 24-X₆-SEQ ID NO: 25-X₇ -SEQ ID NO: 26-t, where X₁ =K or S; X₂ =S or H; X₃ =R,L, or A; X₄ =L or T; X₅ =M or S; X₆ =N or a deletion; SEQ ID NO 25 is R;X₇ =S or deletion, and t=a carboxy or amidated carboxyterminal group,and where SEQ ID NO: 22 is C K G K G A; SEQ ID NO: 23 is C; SEQ ID NO:24 is Y D C C T G S C; and SEQ ID NO: 26 is G K C.

4. Within Group II, the amino acid variations which occur at the eightnon-conserved residues are allowed, including peptides in which thecarboxy terminus is amidated or has a free acid form. Thus, the secondgroup compound derivatives include the peptide structures having theform: SEQ ID NO: 27-X₁ X₂ X₃ -SEQ ID NO: 28-X₄ -SEQ ID NO: 31-t, whereX₁ =X or R; X₂ =T or L; X₃ =S or M, X₄ =X or P; and t=a carboxy oramidated carboxyterminal group, and where SEQ ID NO: 27 is C L S X G S SC S; SEQ ID NO: 28 is Y N C C R S C N; and SEQ ID NO: 31 is Y S R K C R.

5. Considering both active groups together, amino acid positions whichare conserved in all active species are preserved. Thus, for example,the Cys residues, the 5-position glycine, the 13-position tyrosine, the19-position serine, and the 26-position lysine are all preserved.Preferred OCT analogs or derivatives may be selected by comparing, forpurposes of inter-sequence conservation and substitution, thosesequences known to be active. For example, in the case of the treatmentof pain, omega-conopeptides MVIIA (SNX-111), SNX-239, SNX-199, TVIA(SNX-185) and SNX-236 are known active compounds. Active derivatives arethose peptides having, in addition to the conserved cysteine residuesdescribed above, a conserved glycine residue at position 5, conservedserine residues at positions 9, 19, and 24, and a conserved lysineresidue at position 26. Inter-sequence substitution of variable residuesis then preferable in the formation of active analogs. For example,analog position 2 may be occupied by a lysine or a leucine residue, andposition 6 may be occupied by an alanine or a serine residue.

6. Considering both active groups together, there are amino acidpositions which are likely to be variable within the range of activespecies. For example, the position 2 amino acid may be lysine orleucine, the position-3 amino acid may be glycine or serine, and theposition 4 amino acid, hydroxyproline or arginine. In addition, if thetwo or more amino acids at a variant position are in a commonsubstitution class, substitution within that class may be favorable.Standard substitution classes are the six classes based on common sidechain properties and highest frequency of substitution in homologousproteins in nature, as determined, for example, by a standard Dayhofffrequency exchange matrix (Dayhoff). These classes are Class I: Cys;Class II: Ser, Thr, Pro, Hyp, Ala, and Gly, representing small aliphaticside chains and OH-group side chains; Class III: Asn, Asp, Glu, and Gln,representing neutral and negatively charged side chains capable offorming hydrogen bonds; Class IV: His, Arg, and Lys, representing basicpolar side chains; Class V: Ile, Val, and Leu, representing branchedaliphatic side chains, and Met; and Class VI: Phe, Tyr, and Trp,representing aromatic side chains. In addition, each group may includerelated amino acid analogs, such as ornithine, homoarginine, N-methyllysine, dimethyl lysine, or trimethyl-lysine in class IV, and ahalogenated tyrosine in Group VI. Further, the classes may include bothL and D stereoisomers, although L-amino acids are preferred forsubstitutions.

7. Considering the known inactive species, substitutions to amino acidswhich are present in inactive species, but not active ones, at anyselected residue position, are not favored to preserve activity in theactive compounds. Thus, for example, although a 3-position serine ispresent in both active and inactive compounds, 4-position serine orthreonine is present in inactive species only, and either substitutionis thus disfavored.

The above amino acid selection rules 6-7 are intended as a guide forallowed amino acid substitutions within active omega-conopeptides. Oncean amino acid substitution or modification is made, the peptide isfurther screened for the requisite calcium channel antagonist activity,and the requisite activities for inhibition of neurotransmitter releaseand binding to the appropriate OCT binding site of neuronal membranes,as described above.

Several of the amino acid substitutions or modifications to theomega-conopeptide illustrate the principles outlined above.

Omega-conopeptides which are selected on the basis of these criteria,discussed in detail below, are tested for ability to produce analgesiain a standard test of analgesia, such as the Rat Tail-Flick test,wherein analgesia is measured by a prolongation of reaction time to anoxious radiant heat stimulus.

E. In vitro Properties of Neuropatholytic Omega-Conopeptides

1. Calcium Channel Blocking Activity.

Calcium channel blocking activity was measured electro-physiologicallyin neuronal (N1E-115 or IMR-32) cell lines, as described in Section II.Omega-conopeptides having calcium channel blocking activity are thosewhich block calcium currents in such cell lines with potencies in therange observed for omega-conopeptides MVIIA and GVIA in N1E-115 cells.

2. High Affinity Binding to OCT Binding Sites. Methods for determinationof binding affinity to OCT binding sites are detailed in Examples 2-3,below.

Compounds were tested for their ability to displace binding of SNX-111,SNX-183, or SNX-230 from their respective binding sites (site 1 and site2, as described above). In displacing SNX-111, it was found thatcompounds having analgesic activity, such as OCT MVIIA (SNX-111),SNX-239, SNX-236, SNX-199 SNX-159 and TVIA (SNX-185), have IC₅₀ valuesbetween about 15 and 300 pM, and K; values between about 1 and 100 pM.In contrast inactive compound SNX-183 had an IC₅₀ of greater than 1000pM for binding at the MVIIA site.

From the foregoing, it is seen that active compounds in accordance withthe invention are characterized by a high binding affinity for MVIIAbinding site 1. The binding affinity for these sites may becharacterized as follows. In the first approach, the binding affinity ofthe compound for binding site 1, as estimated by IC₅₀ in displacingMVIIA from the site, is compared directly with those of selected highaffinity active compounds, such as SNX-111 and SNX-185. An activecompound is one whose binding affinity is at least as high as andpreferably within the range of binding affinities measured for such highaffinity OCT's. Secondly, the binding affinity of the test compound canbe characterized by binding to binding site 2, as estimated by IC₅₀ indisplacing MVIIC (SNX-230) or SVIB (SNX-183) from the site. Thirdly, thebinding affinity of the compound can be characterized by the ratio ofbinding constants or relative affinities of the compound for site 1 andsite 2, as just described. Here an active compound is one whose bindingratio is within the range for the selected active peptides, such asMVIIA (SNX-111) and TVIA (SNX-185); i.e., the binding ratio issubstantially within the range of the ratio observed for theomega-conopeptides MVIIA and TVIA.

A number of omega-conopeptide compounds which were tested gave IC₅₀ andK_(i) values lower than or within the ranges of those ofomega-conopeptides MVIIA (SNX-111) and TVIA (SNX-185) for binding at theSNX-111 site, as shown in Table 1, and these compounds should thus beconsidered candidates as analgesic compounds. In addition activecompounds have relatively high selectivities of binding, equivalent orgreater than those of MVIIA and TVIA, as shown in Tables 2 and 3.However, some of these compounds, may not fulfill additional criteriafor analgesic compounds of the invention, as described herein.

3. Inhibition of neurotransmitter release. Another requisite property ofanalgesic OCT compounds, in accordance with the invention, is theirability to specifically inhibit depolarization-evoked andcalcium-dependent neurotransmitter release from neurons. For example, itis shown here that analgesic omega-conopeptides inhibit of electricallystimulated release of acetylcholine at the myenteric plexus of theguinea pig ileum (Example 3). This inhibition is associatedanti-nociceptive activity, as seen in Table 4. Omega-conopeptides havinganalgesic activity have IC₅₀ 's in the range of those values observedfor active omega-conopeptides MVIIA (SNX-111) and TVIA (SNX-185), orless than approximately 50 nM, as observed in this assay.

4. In vivo Measurements of Neuropathic Analgesia.

Relief of neuropathic pain is conveniently measured in one or more of anumber of animal models known in the art, in which an animal's responseto a given pain stimulus is measured following experimental productionof neuropathy. Inhibition of progression of the neuropathic conditioncan also be measured in this model, when measurements are taken inindividual animals over time following the experimental neuropathicinsult. While normal progression of the condition will be expected toresult in animals that are increasingly sensitive to a given painstimulus over time, inhibition of this progression will be observed as aleveling or diminishing of response to pain stimulus with time.

One demonstrated model of neuropathic pain resembles the human conditiontermed causalgia or reflex sympathetic dystrophy (RSD) secondary toinjury of a peripheral nerve. This condition is characterized byhyperesthesia (enhanced sensitivity to a natural stimulus), hyperalgesia(abnormal sensitivity to pain), allodynia (widespread tenderness,characterized by hypersensitivity to tactile stimuli), and spontaneousburning pain. In humans, neuropathic pain tends to be chronic and may bedebilitating. This type of pain is generally considered to benon-responsive or only partially responsive to conventional opioidanalgesic regiments (Jadad, et al., 1992). In accordance with theinvention, neuropatholytic N-type VSCC blocking compounds are effectivein providing relief of neuropathic pain, as described below.

Experiments carried out in support of the present invention wereperformed in a rat model of peripheral neuropathy detailed in Example 4.Briefly, in the model used, rats are subjected to a surgical procedure,described by Kim and Chung (1992) and Bennett and Xie (1988) designed toreproducibly injure peripheral nerves (spinal nerves L5 and L6). Theserats develop a hyperesthetic state, which can be measured, using one ormore paradigms known in the art. Here, mechanical allodynia was measuredby stimulation of neuropathic rat hindlimb using wire hairs havinggraded degrees of stiffness. Analgesic compounds reverse the heightenedsensitivity such animals exhibit to the stimulus.

FIG. 6 shows results in the allodynia test of animals treated withSNX-111 (6A), SNX-239 (6B), SNX-159 (6C) and SNX-230. Data are expressedas percent maximum effect, where the maximum effect indicates a completereversal of surgically induced allodynia, or relative insensitivity tostimulus (maximum equals 15 gram hair stimulus). A baseline of zeroindicates a mean sensitivity to a wire hair graded at less than 3 grams.As shown in FIG. 6A, treatment of rats (n=6/treatment) with 1 or 3 μgSNX-111 resulted in elevation of threshold response. Peak elevation ofresponse due to drug treatment (reversal of allodynia) was observed by30-60 minutes, and effects lasted in excess of 60 minutes. SNX-239showed significant analgesic effects at a dose as low as 0.33 μg, andevoked a prolonged analgesic response of at least 2 hours, as indicated.SNX-159 was also effective against neuropathic pain in this test atsubmicromolar doses (FIG. 6C), while SNX-230was ineffective at suchdoses (FIG. 6D).

FIGS. 7A and 7B show dose response curves derived from the data shown inFIGS. 6A and 6B. These results indicate that analgesic omega conotoxinpeptides, exemplified by SNX-111, are capable of reversing thehyperesthetic effects induced by nerve damage.

F. Dosages and Formulations

From the foregoing, it can be appreciated that treatment with N-channelblocking compounds and, more particularly, omega conopeptides havingbinding and inhibitory activities within the range of activities definedby omega conopeptides MVIIA (SNX-111) and TVIA (SNX-185) are useful inpreventing progression of neuropathic pain. Generally, dosages androutes of administration of the compounds will be determined accordingto the site of the pain and the size of the subject, according tostandard pharmaceutical practices. Intrathecal administration, either asa bolus dosage and as a constant infusion, can be used for treatment andprevention of progression of neuropathic pain. In preferred embodiments,dosages equivalent to at least 0.1-3 μg intrathecal SNX-111 in rats areeffective against peripheral neuropathy of the hindlimb. It is alsoappreciated that compound can be administered perineurally, for exampleby topical or subdermal application to cutaneous regions having affectednerve endings, according to methods known in the art. In addition,administration may by epidural means, as discussed below.

Stabilized formulations, as described in Section I.B above, are usefulin storing and administering therapeutic omega conopeptides according tothe methods described herein. While it may be desirable to neutralizethe solutions prior to administration, formulations utilizing lactate oracidified saline as excipient may also be administered directly viaacute intrathecal bolus injection or by other routes for which acidifiedexcipients are appropriately used. Neutralization, if required, can beaccomplished by dilution into a pharmaceutically acceptable neutralizingexcipient buffer, just prior to injection into the subject. FIG. 9 showsthe results of experiments demonstrating that SNX-111 administered as amethionine-lactate formulation was as effective as SNX-111 alone inreducing neuropathic pain.

For some applications, it may be desirable to include in the omegaconopeptide composition or treatment regimen means for enhancingpermeation of the conopeptide through meningeal tissues which maysurround the damaged or target nerve. Means for enhancing transport ofcompound are known in the art and may include encapsulating theconopeptide in liposomal membranes, addition of a surfactant to thecomposition, addition of an ion-pairing agent, and the like.Alternatively, or in addition, transmeningeal transport may befacilitated by administering to the subject a hypertonic dosing solutioneffective to disrupt meningeal barriers, according to methods well knownin the art. Alternatively, trans meningeal or transcutaneous transportmay be facilitated by modifying the primary sequence ofomega-conopeptide, for example by substituting neutral amino acidsidechains or hydrophobic moieties for cationic residues.

The following examples are intended to illustrate variouscharacteristics of the method of the invention, but are in no wayintended to limit the scope of the invention.

EXAMPLE 1 Preparation of OCT Peptides

Abbreviations used in this example are BOC, tertiary butoxycarbonyl;DCM, dichloromethane; TFA, trifluoroacetic acid; IPM,N-isopropylmorpholine; BOC-AA-OH, BOC amino acid; DIEA,diisopropylethylamine; 2-ClZ, chlorobenzyloxycarbonyl; tosyl,p-toluenesulfonyl; DMF, N,N-dimethylformamide; TFE, trifluoroethanol;SA, symmetrical anhydride of BOC-AA-OH; DCCI,N,N-dicyclohexylcarbodiimide; E, ethyl ether; P, petroleum ether.

Commercially available benzhydrylamine-resin hydrochloride, Lot No.B30101, was obtained from commercial sources (Beckman Instruments Inc.,Palo Alto, Calif. Advanced ChemTech). With this resin, cleavage of apeptide formed on the resin, under the conditions described below,produces a peptide which is amidated at its carboxy end.

A. Preparing Protected Amino Acid Anhydrides Each BOC-AA-OH (2.4 mmol)was dissolved in 5 ml CH₂ Cl₂ and cooled to 0 C. The volume of DCM usedfor BOC-Leu-OH (dried in vacuo) was 12 ml, and the BOC-Leu-OH solutionwas not cooled. 2 ml 0.6 M DCCI in DCM was added and the mixture stirredat 0 C. for 15 min. For BOC-Leu-OH, the mixture was also cooled afterthis addition. Precipitation of N,N-dicyclohexylurea was completed bystorage at -20° C. for 1.5 hour, after which the precipitate wasfiltered and washed with ethyl ether (5 ml). The filtrate was evaporatedto remove solvents and the product was crystallized in the solventsystem given in the Table below. Residual amounts of DCM can affect theexact conditions for crystallization. Recrystallization was performed bydissolving in DCM, evaporating most of the solvent, and recrystallizingfrom the appropriate solvent.

    ______________________________________                                        TABLE OF AMINO ACID SOLVENTS                                                  Amino Acid           Solvent                                                  ______________________________________                                        Ala                  DCM:E:P                                                  Asp (Benzyl)         E:P                                                      Gly                  E:P                                                      Leu                  P                                                        Lys (2-ClZ)          E:P                                                      Met                  E:P                                                      Ser (Benzyl)         E:P                                                      Thr (Benzyl)         E:P                                                      Tyr (2-BrZ)          DCM:P                                                    ______________________________________                                    

B. Preparation of MVIIA

Synthesis of MVIIA peptide was performed on 0.58 g benzhydrilamine resin(0.40 mmol) in a Beckman Model 990 Peptide Synthesizer by a solid-phasemethod based on the primary structure shown in FIG. 1A.

A double coupling protocol was used for the incorporation of residuesCys-25 through Tyr-13, and a triple coupling protocol, for amino acidsMet-12 through Cys-1. Symmetrical anhydrides were used in crystallineform as described in Yamashiro (1987). Crystalline symmetricalanhydrides (1.0 mmole) were each dissolved in 6 ml DCM and stored in theamino acid reservoirs at 4° C. Sidechain protecting groups used were:Cys, 4-MeBenzyl; Lys, 2-ClZ; Ser, Benzyl; Arg, Tosyl; Thr, Benzyl; Asp,Benzyl; Tyr, 2-Br-Benzyl.

Unless specified, volumes were 8 ml, except for step 2 below, which was10 ml, and all reactions were carried out at room temperature. Afterincorporation of the Asp-14 residue, the volume of step 2 was increasedto 15 ml while all other volumes were raised to 10 ml afterincorporation of the Arg-10 residue. The double coupling protocolconsisted of steps 1-16 listed in the Table below.

Amino acids Met-12 through Cys-1 were added by a triple couplingprotocol which included, in addition to steps 1-16, steps 17-20 in theMVIIA protocol Table.

    ______________________________________                                        MVIIA PROTOCOL TABLE                                                          Step       Reagent                                                            ______________________________________                                        1          DCM wash (3 times)                                                 2          67% TFA/M (20 min.)                                                3          DCM wash (2 times)                                                 4          25% dioxane/DCM wash (2 times)                                     5          5% DIEA/DCM wash                                                   6          DCM wash                                                           7          5% DIEA/DCM wash                                                   8          DCM wash (5 times)                                                 9          1.0 mmol SA in DCM (5 min)                                         10         0.5 mmol IPM in 3 ml TFE plus 1 ml DCM                             11         (5 min)                                                            12         0.5 mmol IPM in 5 ml DCM (5 min)                                   13         DMF wash (3 times)                                                 14         1.0 mmol SA in DMF (5 min)                                         15         0.5 mmol IPM in 5 ml DCM (5 min)                                   16         0.5 mmol IPM in 4 ml DMF (5 min)                                   17         DCM wash                                                           18         DCM wash (2 timnes)                                                19         1.0 mmol SA in DCM (5 min)                                                    0.5 mmol IPM in 4 ml DMF (5 min)                                   20         DCM Wash                                                           ______________________________________                                    

Crystalline symmetrical anhydrides (1 mmole) were each dissolved in 6 mlDCM or DMF and stored in the amino acid reservoirs at 4° C. Side-chainprotecting groups used were: Cys, 4-MeBzl; Lys; ClZ; Ser, Bzl; Arg,tosyl; Thr, Bzl; Asp, Bzl; Tyr, BrZ.

For BOC-Arg(tosyl)-OH, the following mixture was prepared: 1.87BOC-Arg(tosyl)-OH, 0.57 g 1-hydroxybenzotriazole, 15 ml DMF, stirred todissolve, cooled to 4° C. added 0.52 ml diisopropylcarbodiimide, andsplit in half for steps 9 and 13. For this coupling, the protocol wasmodified as follows: step 8 was 3 times DCM wash and 2 times DMF wash;step 9 was for 10 min; step 11 was for 10 min; step 13 was for 10 min;step 14 was 0.4 mmol IPM in 4 ml DMF for 10 min; step 15 was for 10 min;step 16 was 1 times DMF wash and 1 time DCM wash. Reaction mixtures insteps 9, 10, 13, 14 and 18 were not drained.

The mixture for a third coupling for incorporating the Arg-10 residueconsisted of 1.00 g BOC-Arg(tosyl)-OH, 1 ml DMF, 5 ml DCM, stirred todissolve, and cooled to 4° C. to which is then added 1.67 ml 0.6M DCCIin DCM.

After the last amino acid had been incorporated, the protected peptideresin was subjected to steps 1-4 to remove the N-terminal BOC group,collected on a filter with use of ethanol, and dried in vacuum to yield2.61 g.

MVIIA has also been successfully synthesized on an ABI 430A synthesizerusing slight modifications of the above protocol.

C. Deblocking and Cleavage in Liguid HF

A mixture of protected peptide resin (1.32 g), 2-mercaptopyridine (0.50g), p-cresol (2.6 g), and liquid hydrogen fluoride (HF) (25 ml) wasstirred at 0° C. for 80 min. The liquid HF was evaporated with a rapidstream of nitrogen gas, first below 0° C. then at 24° C. The mixture wasstirred in ethyl acetate (25 ml) until a finely divided solid wasobtained. The solid was filtered, washed with ethyl acetate, and airdried to yield 1.09 g. This solid was stirred in 50% aqueous acetic acid(10 ml) to dissolve the peptide material, filtered, and washed with 20ml water. The filtrate was freeze-dried to yield 450 mg of fluffypowder.

D. Formation of Disulfide Bridges

A sample (300 mg) of the fluffy powder was dissolved in 30 ml of 0.05Mammonium bicarbonate, 10 mM dithiothreitol (DTT), and 2M guanidinehydrochloride. The solution, which had a pH of 6.7, was allowed to standat 24° C. for 2 hr, then diluted with 120 ml of water and stirred for 20hr at 24° C. DTT (25 mg) was added and the solution allowed to stand at24° C. for 80 min. The mixture was then stirred at 4° C. for 3 days.

E. Isolation of MVIIA OCT

The solution from Part D was acidified with glacial acetic acid (2 ml),evaporated in vacuo to a low volume, and fractionated by gel filtrationon Sephadex G-25 in a 2.5×48 cm column, using 1N acetic acid, to removepeptide polymeric species (exclusion volume), and salts (slowest movingpeak). Fractions (5 ml) were collected, with peptide absorbancemonitored at 280 nm. Fractions corresponding to the monomer peptide werepooled and freeze-dried to give 127 mg of fluffy powder. A sample of themonomeric material (34 mg) was purified by preparative HPLC on a Vydac218 TP1022 column with a gradient of 10-20% acetonitrile in 0.1%trifluoroacetic acid over 50 min at 8 ml/min, with detection at 226 nmand collection of 4 ml fractions. Fractions corresponding to the majorpeak were pooled, evaporated in vacuo to remove acetonitrile, andfreeze-dried to yield 7.7 mg. Analytical HPLC on a Vydac 218 TP104column with the same solvent and gradient over 10 min followed by 10 minof isocratic elution at the 20% composition (1.5 ml/min) gave a singlepeak identical in behavior to an authentic sample of OCT MVIIA. Aminoacid analysis of a 24-hr HCl-hydrolysate gave: Asp, 0.93; Thr,1.05; Ser,2.85; half-cystine, 5.2; Gly, 4.08; Ala, 1.07; Met 0.94; Leu, 1.02;Tyr,0.85; Lys, 3.98; Arg, 2.09.

F. Radio-Iodination of MVIIA MVIIA peptide was iodinated by reactionwith Iodogene™ in the presence of NaI according to Cruz et al., withminor modification. 2 m Ci of carrier-free Na¹²⁵ I, 75 ul 0.5M phosphatebuffer pH 7.4 and 20 ul of 1 ug/ul peptide were added to a polypropylenetest tube coated with 10 ug Iodogenf™. The tube was agitated for 8minutes, and the solution was chromatographed by HPLC through a 10×0.46cm C-8 reverse phase column with a pore size of 300 Å (Brownlee Labs,Santa Clara, Calif.). The sample material was eluted with a gradientfrom 0.1% trifluoroacetic acid to 60% acetonitrile in 0.1%trifluoroacetic acid. The major peak of active radio-iodinated peptidewas resolved at about 2 minutes greater retention time than theunderivatized peptide.

The fractions containing this peak were collected and later diluted foruse in binding experiments. MVIIA, iodinated under the conditions asabove except with non-radioactive NaI, was tested for the ability toinhibit depolarization-dependent ATP release from synaptosomes asdescribed in Ahmad and Miljanich (1988) and found to be as potent inthis regard as the underivatized peptide.

G. Synthesis of Other OCT Peptides

Synthesis of other OCT peptides was according to the solid-phase methoddescribed above, except that a single coupling protocol involving steps1-12 in Part C was used for coupling the first 10 C-terminal amino acidsresidues, and a double coupling method involving steps 1-16, Part C wasused for coupling the final n⁻ 10 N-terminal residues, where n is 24-29.Releasing the peptide from the solid support, removing the blockinggroups, and joining the disulfide bridges were carried out substantiallyas above, or as described in Part H, below. The peptide was separatedfrom salts and polymeric peptide species by gel filtration on SephadexG-25, and purified on preparative HPLC. For binding studies, eachpeptide can be radioiodinated essentially as above.

H. Alternate Oxidation Methods

Two alternative oxidation methods were used in the preparation ofMVIIA/SNX-111.

1. The lyophilized crude linear peptide was dissolved in 3M guanidinehydrochloride and 1.2M ammonium acetate solution to yield aconcentration of approximately 12 mg peptide/ml. DTT was added to aratio of 15 mg DTT per 100 mg peptide, and the mixture was stirred atroom temperature for 1 hour. The solution was diluted 6-fold withdistilled water, and stirred at 4° C. for 3-5 days. The progress ofpeptide oxidation was monitored by HPLC. The endpoint of the oxidationprocess was the complete disappearance of free thiols, determined byEllman reaction.

2. The lyophilized crude linear peptide was dissolved in 3M guanidinehydrochloride and 0.3M potassium phosphate solution to yield aconcentration of approximately 12 mg peptide/ml. After addition of 40 mgcysteine and 15 mg DTT per 100 mg peptide, the pH of the solution wasadjusted to 8.0-8.1 with potassium hydroxide solution. The mixture wasstirred at room temperature for 1 hour. The peptide solution was diluted6-fold with water, and stirred at 40° C. for 3-5 days. The progress ofpeptide oxidation was monitored by HPLC. The endpoint of the oxidationprocess was the complete disappearance of free thiols, determined byEllman reaction. (Method 2 was used in the preparation of SNX-236 andSNX-239).

Following oxidation by either of the above methods, the solution wasacidified with acetic acid to pH 3, and lyophilized.

EXAMPLE 2 Omega-conopeptide Binding to Omega-conopeptide Binding Sitesin Synaptosomal Membranes

A. Preparation of Mammalian-Brain Synaptosomes and SynaptosomalMembranes

Synaptosomes were prepared from rat whole brain or hippocampal region ofbrain. Rats were sacrificed, and forebrains were removed and transferredto 10 ml ice-cold 0.32M sucrose containing the following proteaseinhibitors (PI): 1 mM EGTA; 1 mM EDTA; 1 uM pepstatin; 2 uM leupeptin.Brains were homogenized using a motor-driven Teflon-glass homogenizer(approx. 8 passes at 400 rpm). Homogenates from 4 brains were pooled andcentrifuged at 900×g for 10 minutes at 4° C. Supernatants were thencentrifuged at 8,500×g for 15 minutes. Resulting pellets wereresuspended in 10 ml each ice-cold 0.32M sucrose plus PI with vortexmixing. The suspension was then centrifuged at 8,500×g for 15 minutes.Pellets were resuspended in 20 ml ice-cold 0.32M sucrose plus PI. Thesuspension (5 ml/tube) was layered over a 4-step sucrose densitygradient (7 ml each: 1.2M sucrose, 1.0M sucrose, 0.8M sucrose, 0.6Msucrose; all sucrose solutions containing PI). Gradient tubes werecentrifuged in a swinging bucket rotor at 160,000×g for 60 minutes at 4°C. The 1.0M sucrose layer plus the interface between the 1.0 and 1.2Msucrose layers were collected and diluted with ice cold deionized waterplus PI to yield a final sucrose concentration of 0.32M. The resultingsuspension was centrifuged at 20,000×g for 15 minutes. Pellets were thenresuspended in 5 ml icecold phosphate buffered saline plus PI. Theresulting rat brain synaptosomes were then aliquoted and stored in aliquid nitrogen containment system.

Prior to use in binding assays, synaptosomes were thawed and dilutedwith 3 volumes of ice cold deionized water plus PI. This suspension washomogenized using a PT 10-35 Polytron (setting 6) for two 10-secondbursts. The homogenate was centrifuged at 40,000×g for 20 minutes at 4°C. The resulting pellets were resuspended in about 5 ml of ice coldphosphate buffered saline plus PI. The resulting brain synaptosomalmembrane preparation was aliquoted and stored at -80° C. until use.Protein concentration of the membrane preparation was determined usingBradford reagent (BioRad), with bovine serum albumin as standard.

B. Saturation Binding Assay MVIIA OCT was radiolabeled with ¹²⁵ I-iodineby reaction with Iodogen™, essentially according to the method of Ahmadand Miljanich (1988). Following the Iodogen reaction, the peptidesolution was chromatographed by HPLC through a C-8 reversed phase columnand eluted with a gradient from 0.1% trifluoroacetic acid in water to0.1% trifluoroacetic acid in water/acetonitrile (40:60 vol/vol). Themajor peak of radioactivity following the underivatized MVIIA OCT wascollected.

The binding constant (K_(d)) for ¹²⁵ I!-MVIIA OCT to rat brainsynaptosomal membranes was determined by a saturation binding method inwhich increasing quantities of ¹²⁵ I! MVIIA OCT were added to aliquotsof a synaptosomal membrane preparation (10 ug membrane protein,suspended in binding buffer consisting of 20 mM HEPES, pH 7.0, 75 mMNaCl, 0.1 mM EGTA, 0.1 mM EDTA, 2 μM leupeptin, 0.035 μg/ml aprotinin,and 0.1% bovine serum albumin (BSA), in a total volume of 0.5 ml).Binding at each concentration of labeled compound was determined in theabsence and presence of 1 nM unlabeled MVIIA OCT to determine specificbinding (as described in part B, below). The amount of labeled peptidespecifically bound at each concentration was used to determine B_(max),the concentration of specific binding sites on the synaptosomes, andK_(d), following standard binding analysis methods (Bennett, et al.,1983). Scatchard analysis of saturation binding curve of ¹²⁵ I!MVIIArevealed a K_(d) of about 10 pM for the compound.

B. Competitive Displacement Binding Assay

1. Competitive Displacement of OCT MVIIA.

Rat brain synaptosomal membranes prepared as described in Part A weresuspended in a binding buffer consisting of 20 mM HEPES, pH 7.0, 75 mMNaCl, 0.1 mM EGTA, 0.1 mM EDTA, 2 μM leupeptin, 0.035 μg/ml aprotinin,and 0.1% bovine serum albumin (BSA). ¹²⁵ I!-MVIIA (SNX-111) OCT(25-30,000 cpm, approximately 1500-2000 Ci/mmol) and test compound werealiquoted into polypropylene tubes, in the absence or presence of 1 nMMVIIA (SNX-111) OCT to determine non-specific binding. The membranesuspension was diluted and aliquoted last into the test tubes, such thateach assay tube contained 10 μg membrane protein and the total volumewas 0.5 ml. After incubation for 1 hour at room temperature, tubes wereplaced in an ice bath, then filtered through GF/C filters (Whatman),which were pre-soaked in 0.6% polyethyleneimine and prewashed with washbuffer (20 mM HEPES, pH 7.0, 125 mM NaCl, 0.1% BSA) using a Milliporefiltration system. Just prior to filtration, each assay tube received 3ml ice-cold wash buffer. The filtered membranes were washed with two 3ml volumes of ice-cold wash buffer, dried, and filter-boundradioactivity was measured in a Beckman gamma counter (75% countingefficiency).

Representative displacement binding curves for rat brain synaptosomalmembranes are illustrated in FIG. 3. IC₅₀ values were computed from linefit curves generated by a 4-parameter logistic function. These valuesrepresent the concentration of test compound required to inhibit by 50%the total specific binding of ¹²⁵ I!-MVIIA (SNX-111) OCT to rat brainsynaptosomal membranes, where specific binding is defined as thedifference between binding of ¹²⁵ I!-MVIIA (SNX-111) OCT in the absenceand presence of excess (1 nM) unlabelled MVIIA OCT. Non-specific bindingis that binding of radiolabeled compound which is measured in thepresence of excess unlabeled MVIIA OCT. Such values serve asapproximations of the relative affinities of a series of compounds for aspecific binding site.

2. Competitive Displacement of OCT SVIB.

Rat brain synaptosomal membranes were prepared as described above. OCTSVIB was radiolabeled by iodination with ¹²⁵ I-iodine by the Iodogenreaction. Displacement binding of radiolabeled SVIB on rat brainsynaptosomal membranes was carried out as in Example 4B. SVIBdisplacement curves for several of the omega-conopeptides assayed isshown in FIG. 4. IC₅₀ values and relative potency values were calculatedas described below. Tables 2 and 3 show the relative potency values foromega-conopeptides examined, and the ratio of relative potencies of thecompounds for the OCT MVIIA site and to the SVIB binding site.

The binding constant (K_(i)) for each test substance was calculatedusing non-linear, least-squares regression analysis (Bennett and Xie,1988) of competitive binding data from 2 assays performed in duplicateon separate occasions. The relationship between K_(i) and IC₅₀(concentration at which 50% of labeled compound is displaced by testcompound is expressed by the Cheng-Prusoff equation:

    K.sub.i =IC.sub.50 /(1+ L!/K.sub.d)

where IC₅₀ is the concentration of test substance required to reducespecific binding of labeled ligand by 50% ; L! is the concentration of¹²⁵ I!-MVIIA (SNX-111) OCT used in the experiment; and K_(d) is thebinding constant determined for binding of ¹²⁵ I!-MVIIA (SNX-111) OCT torat brain synaptosomal membranes in saturation binding experiments.Table 3 summarizes computed IC₅₀ for various omega-conopeptides for theMVIIA binding site of rat brain synaptosomal membranes.

Relative potency for displacement of binding is calculated as a ratio ofthe IC₅₀ of the test compound and the IC₅₀ of the reference compound.The reference compound is generally the unlabeled equivalent of thelabeled ligand. Calculation of relative potency is as follows:

     log (relative potency)!=log (IC.sub.50(ref))-log(IC.sub.50(test))

Relative potency values for binding at OCT MVIIA (SNX-111) and OCT SVIB(SNX-183) sites are listed in Table 3.

EXAMPLE 3 Inhibition of Electrically Stimulated Contractions of GuineaPig Ileum

Guinea pigs (300-400 gms) were decapitated and the ileum removed. Asection of ileum about 6 cm from the caecum was placed immediately intoKrebb's modified buffer maintained at 37° C. in a water bath, andaerated with a mixture of 95% O₂ and 5% CO₂. The buffer contains: KCl,4.6 mM; KH₂ PO₄, 1.2 mM; MgSO₄, 1.2 mM; Glucose, 10.0 mM; NaCl 118.2 mM;NaHCO₃, 24.8 mM; CaCl₂, 2.5 mM.

Small pieces of ileum were cut and pulled over a glass pipette, scoredand the longitudinal muscle removed. Each piece was attached to anelectrode at one end and to a force transducer at the other end. Thepreparation was lowered into an organ bath maintained at 37° C. andaerated with O₂ :CO₂. The resting tension was set at 1 gm, and thetissue was stimulated at 30-50 V with a duration of 4.5 msec perstimulation.

Baseline responses (contractions) were recorded for 10-15 min. andaliquots (100 ml) of drug were added to the bath until inhibitionoccurred. Following testing, tissues were washed until original responsemagnitude was achieved.

EXAMPLE 4 Rat Model of Peripheral Neurovathy

Male Sprague-Dawley rats (250-350 gm) were prepared with chronic lumbarintrathecal catheters inserted under halothane anesthesia (Yaksh andRudy, 1976). Animals were placed in a prone position and the leftparaspinal muscles were separated from the spinous processes at the L₄-S₂ levels, as described by Kim and Chung (1992). The left L5and L6nerveroots were exposed and tightly ligated with 6-0 surgical silk suture.Approximately 7-10 days after ligaition, the lumbar subarachnoid spacewas catheterized with saline-filled polyethylene (PE-10) tubing asdescribed by Yaksh and Rudy (1976). The Catheter was anchored with staysutures to the adjacent muscle tissue where it emerged from the cisternamagna. Animals were given at least 3 days to recover before assessingmechanical allodynia thresholds. Allodynia was typically observed tooccur beginning 1-2 days post-surgery and continued for as long as 45days. Animals showing motor deficits were excluded from further study.

For testing, animals were placed in plastic cubicles with open wire meshbottoms. Compound dissolved in preservative-free saline solution wasadministered in a volume of 10 μl through the intrathecal catheter,followed by 10 μl saline to flush the catheter line. Animals were testedfor allodynia at various time points after drug treatment, as describedbelow.

To assess the threshold of a non-noxious stimulus required to produce aleft hind paw withdrawal (allodynia), Von Frey hairs (ranging from0.4-15 grams), were systematically applied to the surgically treatedplantar of the hind paw. The hair was held against the surface withsufficient force to cause slight bending and held for 6-8 seconds.Failure to evoke a response was cause to test the next stiffer hair.Evocation of a brisk withdrawal response was cause to test the nextlower stimulus intensity. This paradigm was repeated according to astatistical method (Dixon, 1976) to define the 50% response threshold.Allodynia was evidenced by a threshold less than 3 grams (referring tothe hair stimulus intensity) exhibited by all surgically treatedanimals.

Results of animals treated with saline, or various doses ofomega-conopeptides are shown in FIG. 6, FIG. 7 and FIG. 8. Data in FIG.6 are expressed as percent maximum effect, where the maximum effectindicates a complete reversal of allodynia, or insensitivity to stimulus(maximum equals 15 gram hair cutoff). A baseline of zero indicates amean sensitivity less than 3 grams. As shown in FIG. 6, treatment ofrats (n=6/treatment) with 1 or 3 μg SNX-111 resulted in elevation ofthreshold response. Peak effects were observed by 30-60 minutes, andeffects lasted in excess of 60 minutes.

FIGS. 8A and 8B show results of tests in which animals were given asingle intrathecal bolus injection of SNX-273 (0.1 μg, 0.3 μg, or 1 μg),SNX-279 (0.1 μg, 0.3 μg, or 1 μg), SNX-111 (0.1 μg) or vehicle (0.9%Sodium Chloride injection, USP, Sanofi Animal Health, Inc., OverlandPark, Kans.). Test control compounds were delivered in a volume of 10 μlfollowed by 10 μl of saline to flush the catheter. Results of allodyniatests, performed as described above, are shown as percentage of maximumpossible effect (MPE):

    %MPE=New Threshold(g)-Baseline Threshold(g)×100/15 grams-Baseline Threshold

According to this analysis, the higher the %MPE, the better theantinociceptive effect. As shown in FIGS. 8A and 8B, SNX-111, SNX-273and SNX-279 each blocked mechanical allodynia significantly incomparison to saline control (asterisks in the figures indicatestatistically significant differences between treatment and saline,p<0.05, Student's t test). The apparent order of potency for suppressionof allodynia is SNX-111=SNX-273>SNX-279. This is consistent with thecompounds' relative affinities at the SNX-111 binding site (IC₅₀ 's:SNX-111, 8 pM; SNX-273, 8 pM; SNX-279, 40 pM).

Animals were also observed for the appearance of general motordysfunction, as evidenced by inability to ambulate symmetrically and forany other overt signs of unusual activity. No effects on motor activitywere observed in saline-treated animals; a dose-dependent tremorcharacteristic of SNX-111 administration was observed in animals givenSNX-111.

EXAMPLE 5 Methionine-Lactate Buffer Formulations

Analgesic efficacy of spinally-administered SNX-111 was tested using amethionine-lactate buffer formulation in the paradigm detailed inExample 4. SNX-111 (10 μg/ml) and L-methionine (50 μg/ml) were dissolvedin a vehicle comprised of sodium lactate (150 mM) adjusted to pH 4-4.5with 250 mM lactic acid. This formulation was used to deliver 0.1 μgSNX-111 intrathecally, as described in Example 5 at 30, 60, 120 and 240minutes after treatment with test or control compound. FIG. 9 showseffects on mechanical allodynia of a single intrathecal bolus injectionof 10 μl saline (open circles) or lactate buffer (150 mM) containing 50μg/ml methionine with (closed squares) or without (closed triangles) 10μg/ml SNX-111. Neither saline alone or methionine lactate control bufferalone was effective to suppress allodynia, whereas the SNX-111formulation was effective in this regard (FIG. 9). Moreover, it wasobserved that %MPE values for saline-treated controls were notsignificantly different from those of animals given methionine-lactatebuffer alone.

Although the invention has been described with respect to particularembodiments, it will be apparent to those skilled that various changesand modifications can be made without departing from the invention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 36                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: MVIIA/SNX- 111, FIGURE 1                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CysLysGlyLysGlyAlaLysCysSerArgLeuMetTyrAspCysCys                              151015                                                                        ThrGlySerCysArgSerGlyLysCys                                                   2025                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: MVIIB/SNX- 159, FIGURE 1                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CysLysGlyLysGlyAlaSerCysHisArgThrSerTyrAspCysCys                              151015                                                                        ThrGlySerCysAsnArgGlyLysCys                                                   2025                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: GVIA/SNX- 124, FIGURE 1                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /note= "where X is hydroxyproline"                     (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /note= "where X is hydroxyproline"                     (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 21                                                              (D) OTHER INFORMATION: /note= "where X is hydroxyproline"                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CysLysSerXaaGlySerSerCysSerXaaThrSerTyrAsnCysCys                              151015                                                                        ArgSerCysAsnXaaTyrThrLysArgCysTyr                                             2025                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: GVIIA/SNX- 178, FIGURE 1                              (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /note= "where X is hydroxyproline"                     (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "where X is hydroxyproline"                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CysLysSerXaaGlyThrXaaCysSerArgGlyMetArgAspCysCys                              151015                                                                        ThrSerCysLeuLeuTyrSerAsnLysCysArgArgTyr                                       2025                                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: RVIA/SNX- 182, FIGURE 1                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /note= "where X is hydroxyproline"                     (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "where X is hydroxyproline"                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CysLysProXaaGlySerXaaCysArgValSerSerTyrAsnCysCys                              151015                                                                        SerSerCysLysSerTyrAsnLysLysCysGly                                             2025                                                                          (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: SVIA/SNX- 157, FIGURE 1                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "where X is hydroxyproline"                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CysArgSerSerGlySerXaaCysGlyValThrSerIleCysCysGly                              151015                                                                        ArgCysTyrArgGlyLysCysThr                                                      20                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: TVIA/SNX- 185, FIGURE 1                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /note= "where X is hydroxyproline"                     (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /note= "where X is hydroxyproline"                     (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 21                                                              (D) OTHER INFORMATION: /note= "where X is hydroxyproline"                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CysLeuSerXaaGlySerSerCysSerXaaThrSerTyrAsnCysCys                              151015                                                                        ArgSerCysAsnXaaTyrSerArgLysCysArg                                             2025                                                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: SVIB/SNX- 183, FIGURE 1                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CysLysLeuLysGlyGlnSerCysArgLysThrSerTyrAspCysCys                              151015                                                                        SerGlySerCysGlyArgSerGlyLysCys                                                2025                                                                          (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: SNX-190, FIGURE 2                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CysLysGlyAlaGlyAlaLysCysSerArgLeuMetTyrAspCysCys                              151015                                                                        ThrGlySerCysArgSerGlyLysCys                                                   2025                                                                          (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: SNX-191, FIGURE 2                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CysAlaGlyAlaGlyAlaLysCysSerArgLeuMetTyrAspCysCys                              151015                                                                        ThrGlySerCysArgSerGlyLysCys                                                   2025                                                                          (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: SNX-193, FIGURE 2                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CysLysGlyAlaGlyAlaLysCysSerArgLeuMetTyrAspCysCys                              151015                                                                        ThrGlySerCysArgSerGlyLysCysGly                                                2025                                                                          (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: SNX-194, FIGURE 2                                     (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 12                                                              (D) OTHER INFORMATION: /note= "where X is Nle"                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      CysLysGlyAlaGlyAlaLysCysSerArgLeuXaaTyrAspCysCys                              151015                                                                        ThrGlySerCysArgSerGlyLysCys                                                   2025                                                                          (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: SNX-195, FIGURE 2                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CysLysGlyAlaGlyAlaLysCysSerArgLeuXaaTyrAspCysCys                              151015                                                                        ThrGlySerCysArgSerGlyAlaCys                                                   2025                                                                          (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: SNX-196, FIGURE 2                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      AsnCysLysGlyAlaGlyAlaLysCysSerArgLeuXaaTyrAspCys                              151015                                                                        CysThrGlySerCysArgSerGlyAlaCysGly                                             2025                                                                          (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: SNX-197, FIGURE 2                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AsnSerCysLysGlyAlaGlyAlaLysCysSerArgLeuXaaTyrAsp                              151015                                                                        CysCysThrGlySerCysArgSerGlyAlaCys                                             2025                                                                          (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: SNX-198, FIGURE 2                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      CysLysGlyLysGlyAlaLysCysSerArgLeuMetTyrAspCysCys                              151015                                                                        ThrGlySerCysAlaSerGlyLysCys                                                   2025                                                                          (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: SNX-200, FIGURE 2                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      CysLysGlyAlaGlyAlaAlaCysSerArgLeuMetTyrAspCysCys                              151015                                                                        ThrGlySerCysArgSerGlyLysCys                                                   2025                                                                          (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: SNX-201, FIGURE 2                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      CysLysGlyLysGlyAlaLysCysArgLysThrSerTyrAspCysCys                              151015                                                                        ThrGlySerCysArgSerGlyLysCys                                                   2025                                                                          (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: SNX-202, FIGURE 2                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      CysLysLeuLysGlyGlnSerCysSerArgLeuMetTyrAspCysCys                              151015                                                                        SerGlySerCysGlyArgSerGlyLysCys                                                2025                                                                          (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: SNX-207, FIGURE 2                                     (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /note= "where X is hydroxyproline"                     (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 21                                                              (D) OTHER INFORMATION: /note= "where X is hydroxyproline"                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      CysLeuSerXaaGlySerSerCysSerArgLeuMetTyrAsnCysCys                              151015                                                                        ArgSerCysAsnXaaTyrSerArgLysCysArg                                             2025                                                                          (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: SNX-231, FIGURE 2                                     (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note= "where X is hydroxyproline"                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      CysLysGlyLysGlyAlaXaaCysArgLysThrMetTyrAspCysCys                              151015                                                                        SerGlySerCysGlyArgArgGlyLysCys                                                2025                                                                          (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: CONOPEPTIDE GROUP 1 FRAGMENT                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      CysLysGlyLysGlyAla                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: CONOPEPTIDE GROUP 1 FRAGMENT                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      Cys                                                                           (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: CONOPEPTIDE GROUP 1 FRAGMENT                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      TyrAspCysCysThrGlySerCys                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: CONOPEPTIDE GROUP 1 FRAGMENT                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      Arg                                                                           1                                                                             (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: CONOPEPTIDE GROUP 1 FRAGMENT                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      GlyLysCys                                                                     1                                                                             (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: CONOPEPTIDE GROUP 2 FRAGMENT                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /note= "where X is hydroxyproline"                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      CysLeuSerXaaGlySerSerCysSer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: CONOPEPTIDE GROUP 2 FRAGMENT                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      TyrAsnCysCysArgSerCysAsn                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: SNX-230, FIGURE 1                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      CysLysGlyLysGlyAlaProCysArgLysThrMetTyrAspCysCys                              151015                                                                        SerGlySerCysGlyArgArgGlyLysCys                                                2025                                                                          (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: SNX-236, FIGURE 2                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      CysLeuSerXaaGlySerSerCysSerArgLeuMetTyrAsnCysCys                              151015                                                                        ArgSerCysAsnProTyrSerArgLysCysArg                                             2025                                                                          (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: CONOPEPTIDE GROUP 2 FRAGMENT                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      TyrSerArgLysCysArg                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: SNX-239, FIGURE 2                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      CysLysGlyLysGlyAlaLysCysSerLeuLeuMetTyrAspCysCys                              151015                                                                        ThrGlySerCysArgSerGlyLysCys                                                   2025                                                                          (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: SNX-199, FIGURE 2                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      CysLysGlyLysGlyAlaLysCysSerAlaLeuMetTyrAspCysCys                              151015                                                                        ThrGlySerCysArgSerGlyLysCys                                                   2025                                                                          (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: SNX 240, FIGURE 2                                     (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "The cysteine residue                           carries an acetyl group"                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      CysLysGlyLysGlyAlaLysCysSerLeuLeuMetTyrAspCysCys                              151015                                                                        ThrGlySerCysArgSerGlyLysCys                                                   2025                                                                          (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: SNX-273, FIGURE 2                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      CysLysGlyLysGlyAlaLysCysSerArgLeuAlaTyrAspCysCys                              151015                                                                        ThrGlySerCysArgSerGlyLysCys                                                   2025                                                                          (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: SNX-279, FIGURE 2                                     (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 12                                                              (D) OTHER INFORMATION: /note= "where X is sulfoxy-methionine"                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      CysLysGlyLysGlyAlaLysCysSerArgLeuXaaTyrAspCysCys                              151015                                                                        ThrGlySerCysArgSerGlyLysCys                                                   2025                                                                          __________________________________________________________________________

It is claimed:
 1. A stable omega conopeptide formulation comprising anomega conopeptide and an anti-oxidant composition capable of preventingmethionine oxidation.
 2. The formulation of claim 1, wherein theanti-oxidant composition includes a carboxylic acid buffer.
 3. Theformulation of claim 2, wherein the carboxylic acid buffer is lactatebuffer.
 4. The formulation of claim 1, wherein the anti-oxidant ismethionine.
 5. The formulation of claim 1, wherein the anti-oxidantcomposition includes lactate buffer and methionine.
 6. Anomega-conopeptide SNX-273 having the sequence: SEQ ID NO:
 35. 7. Anomega conopeptide SNX-279 having the sequence SEQ ID NO: 36.